This project deals with viral genetic variability, particularly of the envelope gene of human immunodeficiency virus type 1 (HIV-1). The high envelope variability of HIV-1 has important consequences for its biologic properties, including cellular host range, cytopathic effects, virulence, and recognition by the host immune system. The latter consequence is a serious challenge in the development of vaccines. One area of study identifies targets of neutralizing antibodies and determines how envelope gene variability affects neutralization. One way in which this is studied is by immunoselection in vitro with infectious molecular clones of HIV-1 and characterization of resultant mutants able to resist neutralization by the selecting antisera or antibodies. Several mutants have been characterized that identify distinct groups of human neutralizing antibodies of broad specificity, and these all appear to recognize nonlinear conformational structures rather than linear peptides. Studies directed towards the contribution of the third hypervariable region of the envelope (V3) to neutralization by natural human sera have shown that in at least some natural human sera (in contrast to hyperimmune animal sera), the V3 disulfide loop constitutes part of a neutralization epitope that is both conformational and broadly reactive. Data with HIV-1 V3 mutants that resist neutralization with M77, a murine anti-V3 neutralizing antibody, have led to three-dimensional structural models of the V3 loop. The M77 antibody has been cloned and sequenced, and the solution of its tertiary structure will be used to further refine the structure of the V3 model. Antibodies from HIV-1-infected individuals have been molecularly cloned and are being screened for HIV-1 neutralizing antibodies. A panel of cloned neutralizing antibodies will be very useful in understanding the molecular basis for antibody neutralization and precisely how this is affected by envelope variability. Studies on how genetic variation affects cellular host range centers on infectious clones of HIV-I(IIIB) variants which can grow in macrophages or in epithelioid cells, and are identifying the genes responsible for the altered host ranges.